Picornaviruses are small, non-enveloped viruses that package a single copy of a positive-sense, 3' polyadenylated genomic RNA into their icosahedral capsids. The replication of picornaviruses in mammalian cells requires a combination of genetic economy, highly specific macromolecular interactions, and utilization of host cell functions in many steps of the virus infectious cycle. In the experiments outlined in this renewal application, an analysis of such a combination will be brought to bear on the mechanisms of RNA replication in picornavirus-infected cells. Viral RNA replication requires template recognition in a milieu of cytoplasmic mRNAs that are also polyadenylated at their 3' termini. Major determinants of template recognition and the subsequent assembly of active RNA replication complexes are present in genomic RNA sequences and the viral replication proteins they encode. The proposed experiments will attempt to identify the protein-protein interfaces of a key protein component of picornavirus RNA replication (polypeptide 3CD) and how such interfaces may direct functional multimerization with itself or other viral or cellular proteins. A comprehensive approach is outlined to identify the protein requirements specific for poliovirus positive strand RNA synthesis, an approach that will include cell culture assays to complement in vitro experiments. Finally, the proposed studies will establish in vitro RNA replication assays for both human rhinovirus and coxsackievirus as the first steps to identifying host cell specific factors required for RNA synthesis by these picornaviruses. Information gained from these studies should provide new insights into the exquisite specificity of picornavirus RNA replication complexes and how host cell factors are utilized in the formation of such complexes. Understanding the basis of viral-specific functions may ultimately lead to defining novel targets for chemotherapeutic intervention during viral infections of humans. [unreadable] [unreadable] [unreadable]